Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease
Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease
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DOI：
10.1016/j.jviromet.2015.05.014
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发表时间：
2015-09-15
影响因子：
3.1
通讯作者：
Dunowska, Magdalena
中科院分区：
文献类型：
--
作者：
Giles, Julia C.;Perrott, Matthew R.;Dunowska, Magdalena
本研究的目的是建立一种分离系统，用于分离一种最近被描述的、迄今未被培养的、与负鼠神经系统疾病（称为摇摆负鼠病（WPD））相关的有袋类尼尼病毒。以成年澳洲帚尾负鼠（Trichosurus vulpecula）肝脏为材料，建立了负鼠巨噬细胞原代培养物。在病毒传代至第5代的感染细胞培养液中检测到高病毒拷贝数（高达6.9 x 10(8)/mL细胞裂解液），表明假定的WPD病毒（WPDV）正在培养细胞中复制。采用碘二醇密度梯度超离心法制备密度为1.09 g/mL的纯化病毒原液。在纯化病毒的阴性染色制备中，用电子显微镜观察到直径约60 nm的病毒样颗粒。WPDV在巨噬细胞培养中的一步生长曲线显示，感染后6 ~ 12 h细胞内病毒RNA增加最多。在感染后24小时检测到细胞相关病毒RNA的最高水平，随后下降。细胞外RNA水平在感染后9小时开始升高，在感染后48小时检测到最高水平。WPDV体外培养系统的建立将有助于进一步表征这种新型尼多病毒。(c)2015 Elsevier B.V.版权所有
The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9 x 10(8)/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12 h post-infection. Maximum levels of cell-associated viral RNA were detected at 24 h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9 h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus. (C.) 2015 Elsevier B.V. All rights reserved.